Protein poly (ADP-ribosyl) ation (PARylation) is an important posttranslational modification that regulates cell-survival and cell-death programs, and is being recognized as playing a role in an increasing number of other biological functions. Recent observations suggest that, in some cases, PARylation of a protein serves as a signal for its ubiquitination and degradation. Axin is the first example in which the PARylation-dependent ubiquitination and degradation has been clearly demonstrated. Our collaborators have recently identified RNF146 as a key protein for ubiquitinating PARylated Axin. RNF146 contains only two conserved domains, a RING domain that is likely an E3 ubiquitin ligase, and a WWE domain with unknown function. Now our biochemical analyses demonstrate that the RNF146 WWE domain is a specific poly (ADP- ribose) (PAR) binding domain. Strikingly, most WWE-domain containing proteins also contain an E3 ubiquitin ligase domain (either a RING or a HECT domain). This suggests that there may be a family of E3 ubiquitin ligases that use their WWE domains to specifically recognize PARylated proteins as their ubiquitination substrates. In this proposal, we have two major goals. The first is to further characterize the specific RNF146 WWE-PAR interaction, to reveal the structural basis of this recognition through structural determination, and to analyze the molecular mechanism underlying PARylation-dependent ubiquitination by RNF146. These studies will provide a paradigm for understanding protein PARylation and PARylation-dependent ubiquitination. The second goal is to determine crystal structures of poly (ADP-ribose) glycohydrolase (PARG), the only known enzyme responsible for PAR degradation in the cell and a drug target for cancer and other diseases, and its complexes with the PARG substrate and inhibitors. This line of research will be critical not only for understanding the molecular mechanism of PARG catalysis, regulation, but also for developing better PARG inhibitors. The availability of specific PARG inhibitors will have a significant impact on efforts to explore the biological role of protein PARylation in addition to their potential therapeutic value. PUBLIC HEALTH RELEVANCE: We will focus on the structural and biochemical analysis of protein PARylation and PARylation- dependent ubiquitination, which is crucial for many biological processes. We will also work on the structure and regulation of PARG, a key enzyme regulating protein PARylation. PARG inhibitors may be useful for preventing ischemic brain injury and other therapeutic purposes.